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Pseudoxanthomonas Wuyuanensis Sp. Nov., Isolated from Saline-Alkali Soil

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Pseudoxanthomonas Wuyuanensis Sp. Nov., Isolated from Saline-Alkali Soil %paper no. ije056796 charlesworth ref: ije056796& New Taxa - Proteobacteria Open Access at HighWire International Journal of Systematic and Evolutionary Microbiology (2014), 64, 000–000 DOI 10.1099/ijs.0.056796-0 Pseudoxanthomonas wuyuanensis sp. nov., isolated from saline-alkali soil Dai Li,1 Huancheng Pang,2 Licui Sun,3 Jinping Fan,1 Yuyi Li2 and Jianli Zhang1 Correspondence 1School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China Jianli Zhang 2Institute of Agri-resources and Regional Planning, CAAS, Beijing 100081, PR China [email protected] 3Department of Nutrition and Metabolism, National Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, PR China A bacterium, designated XC21-2T, was isolated from a saline-alkaline soil sample from China. Cells were Gram-stain-negative, rod-shaped and motile and grew optimally at 35–37 6C, pH 6.0–7.0 and in the presence of 0.5 % (w/v) NaCl. Growth occurred in the range pH 5.5–9.0 and in the presence of up to 4 % (w/v) NaCl. The major cellular fatty acids were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 1v9c. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an uncharacterized amino-group-containing polar lipid. The major quinone was ubiquinone 8 (Q-8) and the G+C content of the genomic DNA was 66.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain XC21-2T formed a tight phylogenetic lineage with Pseudoxanthomonas dokdonensis DS-16T within the genus Pseudoxanthomonas and was most closely related to P. dokdonensis DS-16T and P. mexicana AMX 26BT, with 97.9 and 97.5 % 16S rRNA gene sequence similarity, respectively. On the basis of the unique physiological profile of the isolate and its phylogenetic divergence from known species, strain XC21-2T represents a novel species within the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas wuyuanensis sp. nov. is proposed. The type strain is XC21-2T (5CGMCC 1.10978T5KCTC 23877T). The genus Pseudoxanthomonas was described by Finkmann Strain XC21-2T was isolated from a saline-alkaline soil sample et al. (2000) for Pseudoxanthomonas broegbernensis, repre- [pH 8.5; salt content 0.4 % (w/w)] collected from a sun- sented by a strain isolated from an experimental biofilter flower plot in Wuyuan county (40u 469 300 N107u 359 700 E) ; supplied with the waste gas of an animal-rendering plant. in Inner Mongolia in north-west China. The soil sample was At the time of writing, the genus contained 14 species, which suspended in distilled water and then the sus- were isolated from a biofilter (Finkmann et al., 2000), a hot pension was diluted with sterile distilled water and incu- spring (Chen et al., 2002), soils (Thierry et al., 2004; Chang bated at 35 uC on Petri dishes containing nutrient agar (3 g et al., 2005; Yang et al., 2005; Harada et al., 2006; Yoo et al., beef extract, 5 g peptone, 5 g NaCl, 1 l distilled water; 2007; Young et al., 2007; Lee et al., 2008; Kumari et al., 2011) pH 7.6) for 3 days. Purification was obtained by streaking and compost (Weon et al., 2006). Members of the genus single colonies on Petri dishes filled with trypticase soy agar Pseudoxanthomonas are Gram-stain-negative rods and con- (TSA; Difco); purified strains were maintained on TSA at tain ubiquinone 8 (Q-8) as the predominant isoprenoid 4 uC and as glycerol suspensions (20 %, v/v) at 220 uC. quinone. The genomic DNA G+C content is 65–70 mol%. Cells for morphological, physiological and biochemical The cellular fatty acids are of the iso-/anteiso-branched type, characterization of strain XC21-2T and reference strains with iso-C normally predominating and iso-C 3-OH 15 : 0 13 : 0 were prepared by routine cultivation on TSA at 35 uC. Cell always absent with one exception; Pseudoxanthomonas morphology and motility were deduced from direct spadix contains iso-C 3-OH (Thierry et al., 2004; Lee 13 : 0 observations of fresh cultures using light microscopy et al., 2008). (BH-2; Olympus) and transmission electron microscopy (JEM-1400; JEOL). Growth at 4–45 uC, pH 5.0–10.0 (in The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene increments of 0.5 pH units) and 0–5 % (w/v) NaCl (in sequence of strain XC21-2T is JN247803. 1.0 % increments) was examined on TSA. Gram staining Two supplementary figures are available with the online version of this was performed as described by Gerhardt et al. (1981). paper. Activities of catalase and oxidase and hydrolysis of Tween 056796 G 2014 IUMS Printed in Great Britain 1 %paper no. ije056796 charlesworth ref: ije056796& D. Li and others 80, casein, gelatin, starch and urea were investigated as as the spraying reagent for detection of total lipids (Gupta described by Cowan & Steel (1965). Hydrolysis of aesculin et al., 2009; Sharma et al., 2010; Nigam et al., 2010). The T and nitrate reduction were studied as described by La´nyi major fatty acids of strain XC21-2 (.10 %) were iso-C15 : 0, (1987). Sensitivity to antibiotics was tested on TSA plates iso-C16 : 0 and iso-C17 : 1v9c; methyl-branched fatty acids using antibiotic discs. Additional enzyme activities and corresponded to about 80 % of the total cellular fatty acids biochemical characteristics were determined by using the (Table 2). The absence of the fatty acid iso-C13 : 0 3-OH is a API ZYM (37 uC; 4 h) and API 20NE (30 uC; 24 h) kits, as general property of species of the genus Pseudoxanthomonas, described by the manufacturer (bioMe´rieux). The results T but strain XC21-2 had iso-C13 : 0 3-OH in trace amounts, as of phenotypic and physiological characterization of strain T T reported for P. broegbernensis DSM 12573 , P. suwonensis XC21-2 and type strains of related species are given in T T 4M1 and P. spadix IMMIB AFH-5 . Q-8 was the predo- Table 1 and the species description. minant ubiquinone of strain XC21-2T, in agreement with Cells for analysis of cellular fatty acids were cultivated the characteristics of species of the genus Pseudoxanthomonas. on TSA at 30 uC for 48 h. Fatty acid methyl esters were Major polar lipids present in strain XC21-2T were extracted and prepared according to the standard protocol phosphatidylethanolamine, phosphatidylglycerol, dipho- of the MIDI/Hewlett Packard Microbial Identification sphatidylglycerol and an uncharacterized amino-group- System (Sasser, 1990). Isoprenoid quinones were extracted containing polar lipid (Fig. S1, available in IJSEM according to the method of Komagata & Suzuki (1987) and Online). The major polar lipids of strain XC21-2T were analysed using reversed-phase HPLC and a YMC ODS-A the same as those of Pseudoxanthomonas indica and (25064.6 mm) column. Polar lipid analysis was performed Pseudoxanthomonas sacheonensis. The DNA G+C content by two-dimensional TLC using molybdatophosphoric acid of strain XC21-2T was analysed according to the methods Table 1. Phenotypic characteristics that differentiate strain XC21-2T from type strains of phylogenetically related species Strains: 1, XC21-2T;2,P. dokdonensis DS-16T;3,P. mexicana AMX 26BT; 4, P. daejeonensis TR6-08T (data from Yang et al., 2005); 5, P. > broegbernensis B1616/1T (Thierry et al., 2004); 6, P. japonensis 12-3T (Thierry et al., 2004); 7, P. koreensis T7-09T (Yang et al., 2005); 8, P. suwonensis T T 4M1 (Weon et al., 2006); 9, P. yeongjuensis GR12-1 (Yoo et al., 2007). +, Positive test or growth; 2, negative test or no growth; V, variable growth between duplicates; W, weak response; ND, no data available; ONPG, o-nitrophenyl b-D-galactopyranoside. All strains are negative for assimilation of D-mannitol, D-gluconate, caprate, adipate and phenylacetate. Data were obtained from this study unless indicated. Characteristic 1 2 3 4 5 6 7 8 9 Motility and flagellum + 2 ++ ++2 ++ Optimum temperature (uC) 35–37 30 30–37 30 30 30–37 30 30 28 Growth at: 10 uC ++ + W ++ W ++ 42 uC W 22 2 2 2 2 + 2 Catalase ++ W ++2 +++ Arginine dihydrolase 22 2 2 2 2 + 22 Urease 22 2 2 2 2 + 22 Hydrolysis of: Casein + 2 ++ 2 ++ 2 + Gelatin ++ + 22+ 22+ Tween 80 ++ + ND ++ND 2 ND Starch 22 VND 22ND 2 ND Aesculin ++ + + + + 2 ++ ONPG + 22 +++2 ++ Assimilation of: D-Glucose ++ + + + + 2 ++ L-Arabinose + 22 ++22 + 2 D-Mannose 2 ++ 2 + 22 22 N-Acetylglucosamine ++ + + + + 2 ++ Malate + 22 2 2+ (L)* 222 Citrate 22 2 2 + 22 22 Nitrate reduction 2 + 22 222 + 2 DNA G+C content (mol%) 66.2 65.1aD 67.8±2b 68.7±0.4 66.5±0.8 65.2±1 69.5±0.5 67.5±0.9 63.4 *Positive for assimilation of L-malate. DData taken from: a, Yoon et al. (2006); b, Thierry et al. (2004). 2 International Journal of Systematic and Evolutionary Microbiology 64 %paper no. ije056796 charlesworth ref: ije056796& Pseudoxanthomonas wuyuanensis sp. nov. of Tamaoka & Komagata (1984) and was determined to be calculated according to Kimura (1980). DNA–DNA 66.2 mol%, which is similar to results reported for other hybridization was carried out to evaluate genomic DNA members of the genus Pseudoxanthomonas (Table 1). relatedness between strain XC21-2T and its closest neigh- bours (P. dokdonensis DS-16T and P. mexicana AMX 26BT) < Genomic DNA was prepared from strain XC21-2T using a thermal denaturation procedure (De Ley et al., according to Yoon et al. (1996). The 16S rRNA gene was 1970; Huss et al., 1983) with a UV-1206 spectrophotometer amplified by PCR as described by Yoon et al.
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